Cybernetic models based on lumped elementary modes accurately predict strain‐specific metabolic function

In a recent article, Song and Ramkrishna (Song and Ramkrishna [2010]. Biotechnol Bioeng 106(2):271–284) proposed a lumped hybrid cybernetic model (L‐HCM) towards extracting maximum information about metabolic function from a minimum of data. This approach views the total uptake flux as distributed among lumped elementary modes (L‐EMs) so as to maximize a prescribed metabolic objective such as growth or uptake rate. L‐EM is computed as a weighted average of EMs where the weights are related to the yields of vital products (i.e., biomass and ATP). In this article, we further enhance the predictive power of L‐HCMs through modifications in lumping weights with additional parameters that can be tuned with data viewed to be critical. The resulting model is able to make predictions of diverse metabolic behaviors varying greatly with strain types as evidenced from case studies of anaerobic growth of various Escherichia coli strains. Incorporation of the new lumping formula into L‐HCM remarkably improves model predictions with a few critical data, thus presenting L‐HCM as a dynamic tool as being not only qualitatively correct but also quantitatively accurate. Biotechnol. Bioeng. 2011; 108:127–140. © 2010 Wiley Periodicals, Inc.     

Prediction of dynamic metabolic behavior of Pediococcus pentosaceus producing lactic acid from lignocellulosic sugars

A dynamic metabolic model is presented for Pediococcus pentosaceus producing lactic acid from lignocellulose-derived mixed sugars including glucose, mannose, galactose, arabinose, and xylose. Depending on the pairs of mixed sugars, P. pentosaceus exhibits diverse (i.e., sequential, simultaneous or mixed) consumption patterns. This regulatory behavior of P. pentosaceus is portrayed using the hybrid cybernetic model (HCM) framework which views elementary modes of the network as metabolic options dynamically modulated. Comprehensive data are collected for model identification and validation through fermentation experiments involving single substrates and various combinations of mixed sugars. Most sugars are metabolized rather sequentially while co-consumption of galactose and arabinose is observed. It is demonstrated that the developed HCM successfully predicts mixed sugar data based on the parameters identified mostly from single substrate data only. Further, we discuss the potential of HCMs as a tool for predicting intracellular flux distribution with comparison with flux balance analysis.     

Integrating cybernetic modeling with pathway analysis provides a dynamic, systems-level description of metabolic control

Cybernetic modeling strives to uncover the inbuilt regulatory programs of biological systems and leverage them toward computational prediction of metabolic dynamics. Because of its focus on incorporating the global aims of metabolism, cybernetic modeling provides a systems-oriented approach for describing regulatory inputs and inferring the impact of regulation within biochemical networks. Combining cybernetic control laws with concepts from metabolic pathway analysis has culminated in a systematic strategy for constructing cybernetic models, which was previously lacking. The newly devised framework relies upon the simultaneous application of local controls that maximize the net flux through each elementary flux mode and global controls that modulate the activities of these modes to optimize the overall nutritional state of the cell. The modeling concepts are illustrated using a simple linear pathway and a larger network representing anaerobic E. coli central metabolism. The E. coli model successfully describes the metabolic shift that occurs upon deleting the pta-ackA operon that is responsible for fermentative acetate production. The model also furnishes predictions that are consistent with experimental results obtained from additional knockout strains as well as strains expressing heterologous genes. Because of the stabilizing influence of the included control variables, the resulting cybernetic models are more robust and reliable than their predecessors in simulating the network response to imposed genetic and environmental perturbations.     

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

1,2,4‐Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4‐butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2‐ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2‐keto‐3‐deoxy‐xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2‐ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

UV reflectance of inter-subspecific hybrid females obtained by crossing cabbage butterflies from Japan (Pieris rapae crucivora) with those from New Zealand (P. rapae rapae)

Cross-mating was performed between subspecies of cabbage butterfly Pieris rapae crucivora from Japan and P. rapae rapae from New Zealand, whose females are known to reflect and absorb ultraviolet (UV) light, respectively. In the female F ₁ hybrids UV reflectance was intermediate between the two parent females. Further, the UV reflectance was significantly higher in the female F ₁ hybrids derived from females of the New Zealand subspecies than in those derived from females of the Japanese subspecies.     

Calibration of hybrid species distribution models: the value of general‐purpose vs. targeted monitoring data

Aim Temporally replicated observations are essential for the calibration and validation of species distribution models (SDMs) aiming at making temporal extrapolations. We study here the usefulness of a general‐purpose monitoring programme for the calibration of hybrid SDMs. As a benchmark case, we take the calibration with data from a monitoring programme that specifically surveys those areas where environmental changes expected to be relevant occur. Location Catalonia, north‐east of Spain. Methods We modelled the distribution changes of twelve open‐habitat bird species in landscapes whose dynamics are driven by fire and forest regeneration. We developed hybrid SDMs combining correlative habitat suitability with mechanistic occupancy models. We used observations from two monitoring programmes to provide maximum‐likelihood estimates for spread parameters: a common breeding bird survey (CBS) and a programme specifically designed to monitor bird communities within areas affected by wildfires (DINDIS). Results Both calibration with CBS and DINDIS data yielded sound spread parameter estimates and range dynamics that suggested dispersal limitations. However, compared to calibration with DINDIS data, calibration with CBS data leads to biased estimates of spread distance for seven species and to a higher degree of uncertainty in predicted range dynamics for six species. Main conclusions We have shown that available monitoring data can be used in the calibration of the mechanistic component of hybrid SDMs. However, if the dynamics of the target species occur within areas not well covered, general‐purpose monitoring data can lead to biased and inaccurate parameter estimates. To determine the potential usefulness of a given monitoring data set for the calibration of the mechanistic component of a hybrid SDM, we recommend quantifying the number of surveyed sites that are predicted to undergo habitat suitability changes.     

A natural hybrid of Leuciscus cephalus (L.) and Chalcalburnus chalcoides (Güldenstädt)(Osteichthyes‐Cyprinidae) from Lake Tödürge (Sivas, Turkey)

Hybridization between Leuciscus cephalus and Chalcalburnus chalcoides was identified from biometrical features in 55 fish caught from Lake Tödürge, Sivas, Turkey, between April 1994 and November 1997. The hybrid index V _h for metric and meristic characters was intermediate ( V _h = 45·6) compared with their parents ( L. cephalus V _h = 0 or negative and C. chalcoides V _h ≥ 100). Morphological, metric and meristic features of L. cephalus  ×  C. chalcoides also resembled those of the parents. The majority presented an intermediate position which was best shown by the shape of mouth, caudal fin, gill rakers and pharyngeal teeth, and also by the caudal peduncle depth, head and interorbital width, predorsal and preanal distance, height of anal fin, length of anal base and other characters.     

Overproduction of L‐tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale‐up

L‐tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of E. coli was used to overproduce L‐tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimized anthranilic acid feeding into a dissolved oxygen‐stat (DO‐stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L‐tryptophan yield reached 0.211 g/g, 62.3% higher than the previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L‐tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale‐up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L‐tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess.

     

A conditional two‐hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post‐translational modification

The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two‐hybrid (C2H) system, in which bait and prey proteins can be co‐expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co‐expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S‐tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.     

Diploid hybrid origin of Ostryopsis intermedia (Betulaceae) in the Qinghai‐Tibet Plateau triggered by Quaternary climate change

Despite the well‐known effects that Quaternary climate oscillations had on shaping intraspecific diversity, their role in driving homoploid hybrid speciation is less clear. Here, we examine their importance in the putative homoploid hybrid origin and evolution of Ostryopsis intermedia, a diploid species occurring in the Qinghai‐Tibet Plateau (QTP), a biodiversity hotspot. We investigated interspecific relationships between this species and its only other congeners, O. davidiana and O. nobilis, based on four sets of nuclear and chloroplast population genetic data and tested alternative speciation hypotheses. All nuclear data distinguished the three species clearly and supported a close relationship between O. intermedia and the disjunctly distributed O. davidiana. Chloroplast DNA sequence variation identified two tentative lineages, which distinguished O. intermedia from O. davidiana; however, both were present in O. nobilis. Admixture analyses of genetic polymorphisms at 20 SSR loci and sequence variation at 11 nuclear loci and approximate Bayesian computation (ABC) tests supported the hypothesis that O. intermedia originated by homoploid hybrid speciation from O. davidiana and O. nobilis. We further estimated that O. davidiana and O. nobilis diverged 6–11 Ma, while O. intermedia originated 0.5–1.2 Ma when O. davidiana is believed to have migrated southward, contacted and hybridized with O. nobilis possibly during the largest Quaternary glaciation that occurred in this region. Our findings highlight the importance of Quaternary climate change in the QTP in causing hybrid speciation in this important biodiversity hotspot.     

A simple photograph‐based approach for discriminating between free‐ranging long‐finned (Globicephala melas) and short‐finned (G. macrorhynchus) pilot whales off the east coast of the United States

Line transect based abundance estimation is complicated for long‐finned (LFPW, Globicephala melas) and short‐finned (SFPW, G. macrorhynchus) pilot whales because of their similarity in appearance and their overlapping summertime range in some areas. We developed a photograph‐based approach to distinguish between species of free‐ranging pilot whales in the northwest Atlantic. We collected skin samples and photographs during the summers of 2004–2007 and used skin samples to distinguish species based on mitochondrial DNA. Relative morphometric measurements from photographs were examined using mixed‐effect models and logistic regression. The best model among 94 candidate models had an overall classification error rate of 2.5%. We tested the presence/absence of pigmentation in four regions of the dorsal body (melon, eye, cape, and saddle) for differences. Pigmentation was present in all four regions in 100% of the SFPWs sampled. Melon patch, blaze, and saddle patch pigmentation were present in 6%, 68%, and 50%, respectively, of the LFPWs, but the cape was completely absent. Both types of analyses provided positive species discrimination of free‐ranging animals. We created a cost‐effective, simple tool which could ultimately assist in providing appropriate management, mitigation, and conservation strategies for both northwest Atlantic species of pilot whales.     

The effect of marker choice on estimated levels of introgression across an avian (Pipridae: Manacus) hybrid zone

Hybrid zones are often characterized by narrow, coincident clines for diverse traits, suggesting that little introgression occurs across them. However, this pattern may result from a bias in focussing on traits that are diagnostic of parental populations. Such choice of highly differentiated traits may cause us to overlook differential introgression in nondiagnostic traits and to distort our perception of hybrid zones. We tested this hypothesis in an avian hybrid zone by comparing cline structure in two sets of molecular markers: isozyme and restriction fragment length polymorphism markers chosen for differentiation between parental forms, and microsatellite markers chosen for polymorphism. Two cline‐fitting methods showed that cline centre positions of microsatellite alleles were more variable than those of isozyme and restriction fragment length polymorphism markers, and several were significantly shifted from those of the diagnostic markers. Cline widths of microsatellite alleles were also variable and two‐ to eightfold wider than those of the diagnostic markers. These patterns are consistent with the idea that markers chosen for differentiation are more likely to be under purifying selection, and studies focussed on these markers will underestimate overall introgression across hybrid zones. Our results suggest that neutral and positively selected alleles may introgress freely across many hybrid zones without altering perceived boundaries between hybridizing forms.     

Analysis of non‐Saccharomyces yeast populations isolated from grape musts from Sicily (Italy)

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.